ProjectSummary/Abstract There is an urgent clinical need to understand and distinguish indolent from aggressive prostate cancers to inform treatment. The thesis work to date revealed DNA hypermethylation that distinguishes indolent from aggressive prostate cancer. DNA hypermethylation in promoters correlates with reduced expression in aggressivediseaseforgeneswherereducedexpressioncorrelateswithpooroutcomes.However,themajority of alterations occur outside promoters and are enriched for enhancer-like chromatin. The hypothesis of this proposalisthatdifferentiallymethylatedregions(DMRs)innon-promoterregionsfunctiontoregulateenhancer andinsulatorelementswhichincreasestumoraggressivenessbychangesinoncogeneandtumorsuppressor expression within topologically associated chromatin domains. The F99 phase will perform a computational analysis to identify aggressiveness-associated DMRs which occur inside or at the borders of topologically associateddomains(TADs)thatcontainaberrantlyexpressedgenesinaggressivedisease.Usingexistingdata, DMRs within TADs will be classified as enhancer-associated or insulator-associated through integration with histonemodificationandCTCFChIP-seqdatafromprostatecancertissuesandcelllines.Initialexperimental verification of enhancer function will be performed by luciferase reporters. Using prostate cancer cell lines, chromatin conformation capture (3C) will be used to confirm the existence of chromatin loops between the putative enhancer and target gene(s). Finally, genome editing will be performed to delete key motifs in the enhancer.TheeffectongeneexpressionofthetargetwillbeanalyzedwithqPCR,andassayswillbeperformed to measure changes in proliferation and aggressive cellular behavior. For the K00 phase, the applicant will identify mentorship to extend this approach into other cancer types, types of epigenetic modifications, and/or modelsystems(suchasorganoidsandxenografts)andgeneratenewdatausinggenome-wideconformation capture assays (such as capture Hi-C) and histone mark/transcription factor ChIP-seq to inform the interplay betweenthesephenomenaandcancerDMRs.ThiswillestablishnovelfunctionalactivitiesofDNAmethylation incancerandconnectthesechangestogeneexpressionandaggressivenesstoaugmentourunderstandingof thespecificmechanismsofhowcanceraggressivenessisregulatedbyDNAmethylation,leadingtopotential testsandtreatmenttargetstomodifythecourseofthisdiseaseandimprovediagnosticandtreatmentoutcomes.